Lewin A S, Hines V, Small G M
Department of Immunology and Medical Microbiology, University of Florida College of Medicine, Gainesville 32610.
Mol Cell Biol. 1990 Apr;10(4):1399-405. doi: 10.1128/mcb.10.4.1399-1405.1990.
The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.
CIT2基因的产物在其羧基末端有三肽SKL。该氨基酸序列已被证明在哺乳动物细胞中作为过氧化物酶体靶向信号。我们研究了这种线粒体外柠檬酸合酶的亚细胞定位。酿酒酵母细胞在油酸培养基上生长以诱导过氧化物酶体增殖。通过在密度梯度上沉降分析含有膜封闭囊泡和细胞器的部分。在野生型细胞中，柠檬酸合酶活性的主要峰在线粒体部分中回收，但第二个活性峰与过氧化物酶体共沉降。过氧化物酶体活性而非线粒体活性在pH 8.1下孵育时受到抑制，这是由CIT2基因编码的线粒体外柠檬酸合酶的一个特征。在编码线粒体柠檬酸合酶的CIT1基因被破坏的菌株中，柠檬酸合酶活性的主要峰是过氧化物酶体的，并且所有活性在pH 8.1下孵育时都敏感。携带cit2破坏的酵母细胞不能动员储存的脂质，并且在油酸中不形成稳定的过氧化物酶体。我们得出结论，CIT2编码的柠檬酸合酶是过氧化物酶体的，并参与乙醛酸循环。
